Field of Invention
[0001] This invention relates to the detection and treatment, by induced fluorescence and
photochemotherapy, respectively, of certain tissue abnormalities (both cancerous and
non-malignant of endogenous and exogenous origin), hyperproliferative cells, and normal
cells. The invention also relates to the detection and treatment of abnormalities
in body fluids or suspensions of tissues containing abnormal cells by induced fluorescence
and photochemotherapy.
Background of Invention
[0002] Tissue abnormalities involving the skin usually are detected and assessed by a combination
of visual inspection and palpation. In certain clinical situations the sensitivity
of the visual inspection can be enhanced by the use of non-white light (either ultraviolet
or a narrow band in the visible), or by the prior application of a contrast-enhancing
agent such as dilute acetic acid or certain stains. Tissues abnormalities that involve
surfaces that cannot be palpated (such as the bronchi or the urinary bladder) may
be visualized via an appropriate scope. Some specialized scopes can detect induced
fluorescence. If the abnormality in question is associated with a difference in either
the extent or the pattern of tissue vascularization, such a scope may be used to determine
the limits of the area involved by the abnormality, by visualizing an injected bolus
of fluorescein or other fluorescent material as it passes through the vasculature
of both the lesion and the adjacent normal tissue.
[0003] In addition, fluorescence-detecting scopes are being used experimentally to identify
areas of tissue that show strong porphyrin fluorescence following the intravenous
injection of exogenous porphyrins such as hematophorphyrin IX (HpIX), hematoporphyrin
derivative (HpD), or "dihematoporphyrin ether". Such porphyrins tend to accumulate
semi-preferentially in malignant tissues, but they also accumulate in tissues that
are regenerating following an injury or in the rapidly growing tissues of an embryo
or fetus. Normal liver, spleen, and kidney also tend to accumulate these porphyrins.
Using such compounds and fluorescence-detecting scopes, areas of malignant tissue
too small to be identified by standard forms of visual inspection have been identified
in the bronchi and in the urinary bladder.
[0004] Unfortunately, a clinically significant (photosensitizing) amount of porphyrin may
persist in the skin for at least two weeks, (occasionally for more than two months)
following the intravenous injection of HpIX, HpD, or a semi-puridied preparation of
HpD, such as Photofrin II. (Photophrin is a registered trademark of Quadra Logics,
Inc. Vancouver, British Columbia, Canada.) This means that patients must avoid exposure
to sunlight (either direct, or through window glass) for an inconveniently long period
of time post-injection. Understandably, patient compliance often is poor, and accidental
phototoxic "sunburn" is a common occurrence in the weeks following a diagnostic or
therapeutic injection of porphyrin. Persistent photosensitivity is the major hazard
associated with this technique, and is the main reason why it is not used more widely.
[0005] The standard treatments for cancer comprise surgery, radiotherapy and chemotherapy.
However, other forms of treatment are also known, including photochemotherapy or photodynamic
therapy (PDT), based on the discovery made over 90 years ago that unicellular organisms,
.
i.e., certain rapidly growing cells (such as cells of the Lower Kingdom, now referred
to as
Protista), treated with certain chemicals will die when exposed to light. Thus, synthetic
porphyrins have been shown
in vitro to protect cells from infections such as parasites,
e.g., tyromastigotes and sphaeromastigotes of
Tyropanosoma cruzi, J. Parasitol., 75(6) 1989, p. 970-976, and gram positive bacteria, mycoplasma and yeasts,
Malik et al. J. Photochemistry and Photobiology, B. Biology 5 281-293 (1990).
P. acne is known to,
in vitro, produce intracellular protoporphyrin in the presence of exogenous
ALA. Kjeldstad, Conference on Photosensitization and Photochemotherapy of Cancer,
Det Norske Videnskaps-Akademi, March 16-17, 1993, Oslo, Norway.
[0006] PDT is currently being used, on an experimental basis, to treat several different
types of cancer as well as certain non-malignant lesions such as psoriasis. The patient
is given a photo-activatable drug that has some degree of specificity for the tissue
being treated. A tissue volume that includes the target tissue is then exposed to
photoactivating light so as to destroy the target tissue while causing only mild and
reversible damage to the other tissues in the same treatment volume.
[0007] There are two main types of photochemotherapeutic agents in clinical use at present.
The first type, methoxypsoralens, are given systemically. Ultraviolet light is essential
to activate them. Localized exposure of psoralen-containing tissues to ultraviolet
light induces a localized photochemical reaction that causes the drug to bind covalently
to the DNA of living cells, thus destroying their proliferative potential. The second
type, porphyrins and related photosensitizers, are also given systemically (by intravenous
injection), although occasionally they are given either topically or by intralesional
injection. They can be activated by visible (red) light. The localized exposure of
porphyrin-containing tissues to such light ordinarily does not induce a chemical reaction
between cell components and the porphyrin molecules. Instead, the porphyrins act as
catalysts by trapping the energy of the photoactivating light and then passing it
on to molecules of oxygen, which in turn are raised to an excited state that is capable
of oxidizing adjacent molecules or structures. Cell death is not caused primarily
by damage to the DNA, but by damage to essential membrane structures. The goal of
photochemotherapy is sometimes cure (mainly for basal cell carcinomas), but usually
the goal is palliation through local control when none of the standard forms of therapy
are considered likely to offer a significant degree of benefit to the patient.
[0008] Methoxypsoralen (PUVA) therapy is used mainly for the treatment of psoriasis, but
sometimes it is also used to treat very superficial cancers that involve the skin
(mainly mycosis fungoides). However, there are two serious problems with such treatments.
First, the procedure has been demonstrated in humans to be carcinogenic. Second, the
depth at which malignant tissue can be killed is limited to a few millimeters below
the illuminated surface. These problems severely limit the usefulness of the methoxypsoralens
for photochemotherapy.
[0009] 5-Amino-4-oxopentanoic acid, also known as 5-aminolevulinic acid and as δ-aminolevulinic
acid ("ALA") has been described in the cross referenced patents and patent applications
first set forth in this specification for detecting and treating rapidly growing cells.
ALA has also been reported for use in attenuating the growth and killing plants and
insects when applied directly to such organisms followed by exposure to light, based
on work of Rebeiz
et al.
[0010] Synthetic porphyrins have also been used as photochemotherapeutic agents in treating
rapidly growing, e.g. rapidly dividing or rapidly metabolizing infectious cells, such
as infectious pathogens, including protozoal parasites, such as
Plasmodium falciparium (which causes malaria in humans), various other species of
Plasmodia,
Leishmania, and amoebae, pathogenic fungi, and microplasma, including the various parasitic
forms, all such cells and organisms being referred to herein as Protista. The term
Protista as used here and in the literature refers to the lowest orders of the animal and
vegetable kingdoms, single celled or collections of single celled organisms including:
the eukaryotes, including protozoa, fungi and algae, and the prokaryotes, which are
bacteria and blue-green algae.
[0011] At present, the porphyrins most commonly used for photochemotherapy are Hematoporphyrin
IX (HpIX), Hematoporphyrin derivative (HpD) and various semi-purified preparations
of HpD such as commercially available Photofrin
® II, a semi-purified form of HpD. When porphyrins are used as photosensitizers, cell
death results from damage to cell membranes. Consequently, malignant transformation
is not a serious problem. Moreover, since the visible (red) light that is used to
photoactivate porphyrins penetrates tissue much more deeply than does the ultraviolet
light that must be used to photoactivate methoxypsoralens, the depth at which porphyrin-treated
tissue can be killed is substantially greater. Also, since certain types of porphyrins
show a significant tendency to accumulate preferentially in malignant tissues, it
is sometimes possible to destroy malignant tissue without causing clinically significant
damage to adjacent normal tissues.
[0012] The main problem with the systemic use of HpIX, HpD and Photofrin II is that photosensitizing
concentrations persist in the skin for several weeks to several months following their
administration. Consequently, severe accidental phototoxic skin reactions may occur
unless the patient avoids exposure to sunlight (either direct, or filtered through
window glass) until the concentration of the photosensitizer in the skin has been
reduced to a. harmless level. At present, the problem of photosensitivity following
the administration of porphyrins is handled by advising the patient to avoid any form
of exposure to sunlight (or to very bright artificial lights) for a period of at least
two weeks post-injection, and to initiate subsequent exposure to sunlight very cautiously.
Not all patients comply with these instructions, since it often is quite inconvenient
to do so. In addition, the use of a sunscreen with a high blocking factor is recommended
with warning that this will only reduce the hazard somewhat, not eliminate it completely.
In a few cases, patients whose photosensitization persisted for more than a month
post-treatment have been given large daily doses of beta-carotene over a period of
several months in an attempt to prevent accidental phototoxic damage. Finally, attempts
have been made to reduce phototoxicity by applying the photosensitizer topically to
a limited area.
[0013] However, another type of problem is encountered if HpIX or HpD is applied topically
in DMSO (dimethylsulfoxide), Azone, or some other vehicle intended to enhance their
diffusion through tissue. The porphyrins tend to become immobilized wherever they
happened to be when the DMSO or Azone becomes diluted by normal tissue fluids to such
an extent that the porphyrins can no longer diffuse through the tissue (or even remain
in solution). Consequently, the topical application of porphyrins often is associated
with a loss of specificity for malignant tissues, and normal tissues near the site
of application may develop persistent photosensitization from the localized concentration
of porphyrin.
[0014] WO93/20810 published 28 October 1993 discloses medicaments for detecting and treating malignant and non-malignant tissue
abnormalities and lesions of the skin; conjunctiva; respiratory, digestive and vaginal
mucosa; endometrium and urothelium; and for ablating the endometrial tissue and treating
body fluids containing suspended abnormal cells, and for treating cancers of the nervous
system with a photochemotherapeutic method using 5-aminolevulinic acid and precursors
thereof.
[0015] WO95/31189 published 23 November 1995 discloses a medicated wipe comprising an absorbent woven or non-woven fabric, cloth
or tissue substrate, impregnated with a pharmaceutically active agent, wherein the
agent is a substance effective in stimulating melanocytes to produce melanin and/or
is effective in a topical treatment of a skin condition in combination with electromagnetic
radiation falling in the range of 220-700nm.
Obiect of Invention
[0016] This invention relates to a method for the detection of certain types of malignant
and non-malignant cells including a collection of cells, and tissue abnormalities
by induced fluorescence.
[0017] It also relates to a photodynamic (photosynthesizing) treatment method using an agent
which can be administered either systemically or topically which is not in itself
a photosenthesizer but which induces the synthesis or accumulation or both of protoporphyrin
IX (PpIX) and other endogenous porphyrins, their precursors and their photoproducts,
in rapidly growing cells, including abnormal cells in otherwise normal tissues, in
vivo or in vitro.
[0018] The terms porphyrin(s) and their precursors refer to compounds produced in vivo in
the synthesis of heme and other endogenously produced photoactivatable compounds including
their photoproducts.
Summary of Invention
[0019] It is an object of the present invention to provide a precursor of PpIX in the biosynthetic
pathway for heme that can induce synthesis and/or accumulation of PpIX or other endogenous
porphyrins, their precursors and their photoproducts in non-malignant hyperproliferative
lesions, for use in treating of non-malignant hyperproliferative acne lesions of the
skin, following exposure of said lesions to light having a wavelength within the photoactivating
action spectrum of said PpIX to thereby induce photoactivation in said lesions, wherein
the precursor is provided in a solution comprising 20% 5-aminolevulinic acid ("ALA").
[0020] This invention is based on the finding that exogenously administered ALA and other
precursors of PpIX are metabolized in patients to PpIX and that PpIX preferentially
accumulates in rapidly growing cells, as contrasted with less rapidly growing cells.
The rapid growth is correlated with the metabolic activity, so that the differential
accumulation is affected by the relative metabolic activity between dirrerent cells.
[0021] This invention relates to a method for detecting in a patient, a non-malignant lesion
or abnormality which is sensitive to PpIX, namely those which preferentially accumulate
PpIX, comprising administering to said patient an effective amount of a precursor
of PpIX in the biosynthetic pathway for heme so as to induce an accumulation of PpIX
in said lesions, and exposing said lesions to light having a wavelength within the
absorption spectrum of said PpIX, thereby to induce fluorescence in said lesions.
[0022] Another aspect of this invention relates to a method for treating non-malignant hyperproliferative
lesions of the skin which are sensitive to PpIX in a patient, comprising administering
to said patient an effective amount of a precursor of PpIX in the biosynthetic pathway
for heme so as to induce synthesis or accumulation or both of PpIX or other endogenous
porphyrins, their precursors and their photoproducts in said lesions, and exposing
said lesions to light having a wavelength within the photoactivating action spectrum
of said PpIX to thereby induce photoactivation in said lesions.
[0023] Thus, the rapidly growing cells involved can be non-malignant hyperproliferative
lesions. By another aspect this invention relates to the use of a composition comprising
a precursor of protoporphyrin IX in the biosynthetic pathway for heme for the manufacture
of a medicament for treating non-malignant tissue abnormalities and lesions.
[0024] The preferred precursor of protoporphyrin IX is 5-amino-4-oxo-pentanoic acid, otherwise
known as 5-aminolevulinic acid, and a preferred wavelength of the photoactivating
light is in the range of 625 to 670 nm, more preferably a red light of 625 to 640
nm.
Detailed Description of Preferred Embodiment
[0025] Protoporphyrin IX (PpIX), a naturally occurring photosensitizer, is the immediate
precursor of heme in the heme biosynthetic pathway. All nucleated cells have at least
a minimal capacity to synthesize PpIX, since heme is necessary for the synthesis of
various essential heme-containing enzymes. Certain types of cells and tissues can
synthesize relatively large quantities of PpIX. Under normal conditions, the synthesis
of PpIX in such tissues is under such tight feed-back control that the cells produce
it at a rate just sufficient to match their need for heme. However, the usual rate-limiting
step in the process, the synthesis of 5-aminolevulinic acid, can be bypassed by the
provision of exogenous ALA, porphobilinogen, or other precursor of PpIX. Certain tissues
and organs will then accumulate such a large excess of PpIX that they become both
fluorescent and photosensitive. At least in the case of the skin, the PpIX appears
to be synthesized in situ. ALA, which is commercially available from Sigma Chemical
Company and other sources and which is water soluble, can be administered orally,
topically or by injection. The oral and parenteral routes lead to the induction of
clinically useful concentrations of PpIX in certain benign and malignant tissues throughout
the body. Only certain types of tissue synthesize and accumulate clinically useful
amounts of PpIX when provided with an excess of ALA.
[0026] At the present time, treatment of non-malignant hyperproliferative acne lesions of
the skin is contemplated.
[0027] As used herein the term "skin" includes:
(A) the covering of the external surface of most of the body, commonly termed the
skin.
(B) the covering of the external genitalia:
- labia majora, labia minora, clitoris, and associated structures
- glans penis, prepuce, and associated structures
(C) the covering of the zone of transition between skin and the mucosa of the digestive
system:
- anal verge
- vermillion border of the lips
(D) the lining of the external auditory meatus, and the covering of the external surface
of the tympanic membrane
(E) all exocrine glands and associated ducts that are located at least partially within
an epidermal surface described above, or within the underlying dermis, such as the
pilosebaceous units of the skin.
[0028] This invention comprises the administration of ALA, other precursors of PpIX and
other endogenous porphyrins, to the patient. The administration can also be
in vitro as applied to tissues of the patient,
i.e., ex vivo. In
ex vivo methods, tissue containing the rapidly growing cells are removed from the patient,
an effective amount of ALA or endogenous porphyrin is added thereto, then the preparation
is subjected to photoactivating light, before being readministered to the patient.
The amounts of ALA constituting an effective dose can be determined by one skilled
in the art by analogy with the doses used for synthetic porphyrins, based on milligrams
per kilogram body weight for
in vivo systemic application and the typical concentrations for topical or
ex vivo applications. The compound can be conveniently used orally or intravenously at a
dosage of about 10 to 100 mg/kg per single dose, preferedly as a dosage of 40-50 mg/kg;
however split dosages of 10 mg/kg four times per day may also be given. The compound
can be used topically at a dose of between 2% to 100%, with 100% being dry powder.
Ex vivo concentrations of the compound are used on cell suspensions in a range of 1-5mM,
with a preferred range of 1-2mM; however, if serum is present, a higher dose of about
15 mM should be used. If
ex vivo use on whole blood, the compound is used at about 15 mM; however, if an iron kelator,
such as Desferol™ or des ferroxamine, a lower concentration may be used.
[0029] The wavelength of the photoactivating light is of some importance, as it has been
shown that between 1 and 10 percent of incident red light (600-700 nm) can pass through
a slab of human tissue 1 cm thick, whereas only 0.001 percent or less of blue light
(about 400 nm) can pass through the same thickness of human tissue. The photosensitizer
will, therefore, be more successful if it absorbs red light. PpIX does strongly absorb
red light. The present approach has several advantages over the prior art. First endogenous
PpIX has a much shorter half-life in normal tissues (human and mouse, at least) than
does HpIX, HpD or Photofrin
® II. This greatly reduces the danger of accidental phototoxic skin reactions in the
days following treatment. Second, the ALA can be applied topically to certain types
of lesions. This improves the specificity of the treatment, reduces the danger of
accidental phototoxic reactions to a very low level, and greatly reduces the amount
of both ALA and PpIX to which the entire body would be exposed if an equally effective
dose of ALA were to be given systemically.
[0030] Both ALA and PpIX are normal products of metabolism, and are handled quite readily
by the biochemical machinery of the body. However, since very large doses of ALA (like
large doses of HpIX or HpD) are associated with a transient decrease in motor nerve
conduction velocity, it is desirable to reduce the dose of ALA to the minimum that
is still effective. Topical application requires much less ALA than systemic administration.
Third, PpIX is rapidly inactivated by the photoactivating light. Following exposure
of tissues containing PpIX to a therapeutic dose of photoactivating light, there is
a substantial decrease in photosensitization of the tissues within the treatment volume.
Consequently, if PpIX is induced by the topical application of ALA to specific lesions,
the patient can be exposed to sunlight immediately post-treatment without danger of
serious phototoxicity. Also, the dosimetry of the photoactivating light is great simplified.
Fourth, ALA is an effective inducer of PpIX when given by mouth, by topical application,
or by injection. In contrast, HpIX, HpD and Photofrin II are effective in most situations
only when given by injection. The versatility of ALA enhances its acceptability for
routine use by the medical profession, since the oral and topical routes of administration
are much more convenient than the parenteral. Fifth, the normal and abnormal tissues
that can be photosensitized by the administration of ALA are somewhat different from
those that can be photosensitized by the administration of HpIX, HpD or Photofrin
II. Consequently, ALA would be useful in clinical situations in which the other photosensitizers
are not.
[0031] Thus the present technique is not merely another way to do what can be done already
but is; in fact, a significant advance in therapeutic capability.
EXAMPLE 1 ACNE
[0032] Acne is an inflammatory follicular papular and pustular eruption involving the skin.
The treatment of acne using the method of the instant invention would be considered
to be the treatment of either (a) endogenous lesions of the sebaceous apparatus of
the skin due to intrafollicular hyperkeratosis or (b) exogenous bacteria cells present
in the acne lesions, particularly
Propionibacterium (Corynebacterium) acne.
[0033] Evaluation of PpIX induced fluorescence in 8 subjects with mild to moderate truncal
acne was performed. Bacterial infections are frequently associated with lesions of
acne,
e.g., P. acne. Following evaluation of baseline acne lesion fluorescence, ALA solution 10 and 20%
was applied to 10 5 cm
2 sites on the chest or back of volunteers and evaluated at times 0, 3, 8 and 24 hours
after ALA application. One site of each concentration was also occluded with opaque
film for 3 hours and evaluated at similar time points for comparison with unoccluded
sites. Fluorescence of both acneiform lesions as well as surrounding normal skin was
assessed visually using a 4 point grading system (0=none, 4=extremely severe) and
documented photographically.
[0034] In all subjects, unoccluded sites had a gradual increase in PpIX fluorescence that
was dose dependent, maximum at 8 hours, specific for acne lesions and spared normal
surrounding skin. These sites had weak or no fluorescence by 24 hours. Little difference
in fluorescence intensity was noted by lesion type (cornedones vs papules vs pustules)
in the same subject, however, time to maximal fluorescence and maximal fluorescence
intensity was variable from subject to subject. Lesions with surrounding erythema
(larger papules and pustules) developed fluorescence extending to the clinical limit
of erythema. Vehicle control sites remained at baseline. In contrast, occluded sites
developed PpIX fluorescence in both acne lesions and normal surrounding skin that
persisted longer than unoccluded sites and remained present at 24 hours.